Method of anesthesia

ABSTRACT

A method of producing local anesthesia in a mammal experiencing pain in an epithelial tissue region is described. The method includes topically administering to the region, in a suitable pharmaceutical vehicle, an effective dose of a long-acting sodium channel blocking compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/040,903, filed Apr. 2, 1997 and U.S. Provisional Application No.60/076,317, filed Feb. 27, 1998.

FIELD OF THE INVENTION

The present invention relates to a method for producing local anesthesiaby topical administration of sodium channel blocking compounds,including tetrodotoxin and saxitoxin.

BACKGROUND OF THE INVENTION

Pain is a well known phenomenon as an indicator of actual or potentialinjury or tissue damage due to inflammation, ischemia, mechanical orother irritation. Treatment of pain includes the use of localanesthetics, which block neuronal transmission and affect sensation aswell as pain, and analgesics, which relieve pain and additionally mayinterfere with the activity of chemical mediators of inflammation.

Loss or damage of epithelial tissue is usually associated with moderateto severe pain and can result from a number of causes, for example,burns, corneal abrasions, other abnormalities of mucosal tissues, andsurgical procedures involving epithelial and other tissues.

An example of pain associated with a surgical procedure is surgicalcorrection of myopia by excimer laser photorefractive keratectomy.Following photorefractive keratectomy (PRK), patients generallyexperience moderate to severe eye pain in the first 24 to 48 hours.Current pain management with bandage contact lens, non-steroidalanti-inflammatory agents, and oral analgesics mitigates, but does noteliminate, the discomfort in most patients (Cherry, Tutton). Topicalanesthetics have been used to reduce pain, but due to their shortduration of action, frequent administration is required. For example,benoxinate, cocaine, tetracaine, and proparacaine are commonlyprescribed topical anesthetics for management of eye pain. These topicalanesthetics only provide pain relief for short periods, on the order of15 to 30 minutes. Given frequently, these agents can be toxic to thecorneal epithelium and inhibit re-epithelialization (Rosenwasser).

Thus, there is a need in the art for methods of producing long-lasting,local anesthesia without inhibiting re-epithelialization or healing ofother tissues.

SUMMARY OF THE INVENTION

The methods and compositions of the present invention solve, inter alia,the long-recognized need in the art for methods of producing localanesthesia of long duration. In the particular embodiment of producinglong-lasting local anesthesia of the corneal surface of an eye, theinventors have addressed a problem of great clinical significance,showing for the first time that sodium channel blocking compounds, suchas tetrodotoxin and saxitoxin, can produce ocular surface anesthesia oflong duration without impairing re-epithelialization. Moreover, theinventors have shown that the effective doses of those sodium channelblocking compounds have a wide margin of safety and that systemicabsorption of tetrodotoxin topically administered to abraded corneas islow.

The methods and compositions of the invention can be used for anycondition involving ocular surface pain, including local anesthesiafollowing ocular surgery, including PRK, and following injury to theeye. The methods provide significant advantages, including providing atleast 3 hours, preferably at least 4 hours, more preferably at least 6hours, and most preferably, at least 8 hours of local anesthesia withoutaffecting re-epithelialization of the corneal surface (e.g. woundhealing).

The present invention includes methods of producing long-lasting localanesthesia, comprising administering a pharmaceutically acceptablecomposition of a long-acting sodium channel blocking compound, whereinsaid compound binds to the extracellular mouth of the sodium channel,occluding the channel by a mechanism separate from that of localanesthetics, such as proparacaine. Preferably, such methods achievelocal anesthesia of long duration, lasting at least 3 hours (3 to 10hours), preferably at least 4 hours (4-10 hours), and most preferably atleast 6 to 10 hours. Preferred compounds include toxins or analogsthereof that specifically bind to a site formed in part by anextracellular region of the alpha subunit of a sodium channel. Mostpreferred compounds comprise the class of toxins and analogs thatspecifically bind to a site formed by the SS1 and SS2 extracellularregions of the alpha subunit of a sodium channel, wherein such compoundsinclude tetrodotoxin, saxitoxin and analogs thereof. Surprisingly, theselong-acting sodium channel blocking compounds, which are well known,potent neurotoxins, provide long-lasting local anesthesia withoutinhibiting reepithelialization.

Accordingly, it is an object of the invention to provide a method ofproducing local anesthesia in patients experiencing pain associated withdamage to epithelial tissue.

It is another object of the invention to provide a method of producinglocal anesthesia for long-acting pain control.

It is another object of the invention to provide a method of producinglocal anesthesia in epithelial tissues having damage associated withcorneal abrasions, as in, for example, ophthalmic surgery, such aspost-operative photorefractive keratectomy, other forms of cornealrefractive surgery, including excimer laser PRK and LASIK, withoutimpairing healing of the epithelial tissue. Other applications include,but are not limited to, any condition where ocular surface anesthesia oflong duration is desired, including after surgery or injury.

In one aspect, the invention includes a method of producing localanesthesia in a subject experiencing pain in an epithelial tissueregion. The method includes topically administering to the region, in asuitable pharmaceutical vehicle, an effective dose of tetrodotoxin orsaxitoxin.

In one embodiment, the effective dose of tetrodotoxin or saxitoxin isadministered from a formulation containing tetrodotoxin or saxitoxin ata concentration of between 0.001 mM and 10 mM.

In another embodiment, tetrodotoxin or saxitoxin is administered to ade-epithelialized corneal tissue region. For example, tetrodotoxin isadministered to the eye following excimer laser photorefractivekeratectomy by instillation of drops. In this application, tetrodotoxinis typically formulated in a vehicle having a pH of between 4-8, morepreferably between about 5-7.5.

In one embodiment, tetrodotoxin or saxitoxin is administered topicallyevery 6-8 hours for between about 24-72 hours.

The method of the invention, in another embodiment, is for producinglocal anesthesia by topical administration of tetrodotoxin to anepithelial tissue region in the upper or lower gastrointestinal tract.In other embodiments, the epithelial tissue region is associated withgenital lesions in the genital area, with epithelial tissue region is inthe esophagus, or with facial epithelial tissue.

In another aspect, the invention includes a method of producing localanesthesia in the eye of a mammalian subject, by topically administeringto the corneal surface of the eye of the subject, in a pharmaceuticallysuitable vehicle, a pharmaceutically effective dose of tetrodotoxin,saxitoxin or other long-acting sodium channel blocking compound. Suchmethods find application whenever ocular surface anesthesia is desired.

In one embodiment of this aspect, the corneal surface is partially orcompletely de-epithelialized.

In a third aspect, the invention includes a method of reducing painfollowing corneal surgery, including but not limited to excimer laserphotorefractive keratectomy, by topically administering to the cornealsurface of the eye of the subject, in a pharmaceutically suitablevehicle, a pharmaceutically effective dose of tetrodotoxin or saxitoxin.

In one embodiment of the third aspect, the method also includes the stepof instilling drops of a non-steroidal anti-inflammatory compound in theeye of the subject. In other embodiments, the method further includesapplying a bandage contact lens to the eye of the subject, oradministering an antibiotic, steroid, or non-steroidal anti-inflammatorydrug to the subject. Methods of administering combination drops to theeye are also contemplated, wherein such drops contain a compositioncomprising a long-acting sodium channel blocking compound and anantibiotic, steroid, or nonsteroidal anti-inflammatory drug in anopthalmically acceptable vehicle.

In yet another embodiment, the invention includes methods of producinglocal anesthesia in an eye of a mammal, comprising (a) topicallyadministering to the corneal surface of the eye of said mammal, in apharmaceutically suitable vehicle, 0.05%-0.5% proparacaine and (b)topically administering to the corneal surface of the eye of saidmammal, in a pharmaceutically suitable vehicle, an opthalmicallyeffective dose of a long-acting sodium channel blocking compound,wherein step (b) follows step (a). In a preferred embodiment, a 0.05%proparacaine concentration is administered.

Also provided are compositions of a long-acting sodium channel blockingcompound in an opthalmically acceptable pH range of between 4-8, whereinthe concentration of said compound is not greater than 1 mM (0.01 mM to1 mM), preferably not greater than 0.5 mM (0.01 mM to 0.5 mM), morepreferably not greater than 0.2 mM (0.01 mM to 0.2 mM), and mostpreferably not greater than 0.1 mM (0.01 mM to 0.1 mM).

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plot showing corneal blink response in rabbit eyes as afunction of time after administration to the cornea with intactepithelium of 0.1 mM (open circles), 1 mM (open diamonds) and 10 mM(open squares) tetrodotoxin;

FIG. 1B is a plot showing corneal blink response in fellow rabbit eyesfor FIG. 1A following topical administration of the vehicle onlycontrol;

FIG. 2A is a plot showing corneal blink response in rabbit eyes withintact corneal epithelium as a function of time following topicaladministration of 1 mM tetrodotoxin, 10 mM tetrodotoxin or a comparativeanesthetic, 0.5% proparacaine;

FIG. 2B is a plot showing corneal blink response in fellow rabbit eyesfor FIG. 2A following administration of the vehicle only control;

FIGS. 3A-3B are plots showing corneal blink response in rabbit eyesfollowing central corneal epithelial debridement and administration of0.01 mM (open circles), 0.1 mM (open diamonds) or 1.0 mM tetrodotoxin(open squares) (FIG. 3A) and of the fellow eyes treated with a vehicleonly control (FIG. 3B);

FIG. 4 is a plot showing corneal blink response as a function of time inrabbit eyes with central corneal epithelial debridement treated withdoses of 1.0 mM tetrodotoxin administered every 6 hours for 24 hours;and

FIG. 5 is a plot showing the size of a corneal epithelial defect, inmm², as a function of time for untreated rabbit eyes (open squares) andfor rabbit eyes treated with topical 1 mM tetrodotoxin every 8 hours(open diamonds).

FIG. 6 is a chart showing administration of TTX to produce cornealanesthesia of rabbit eyes post-excimer laser keratectomy as measured bya blink response test. Corneal sensation was tested at 3, 6, 9, 12, 15,18, 21, 24, 30, 32 and 40 hours after excimer laser keratectomy. One 40microliter drop of 1 mM TTX or vehicle (control) was administered at 0,6, 12, 18 and 24 hours, as shown by the arrows. The results are graphedas the blink response (mean+/-SEM; N=6).

FIG. 7 is a chart showing the effect of topical administration of TTX oncorneal wound healing following excimer laser keratectomy of rabbiteyes. Wound healing was assessed by measuring the size of the epithelialdefect remaining at 24, 40, 49, 63, 68 and 72 hours after excimer laserkeratectomy. The results are given as the area of the epithelial defectremaining in either the eyes treated with 1 mM TTX or the fellow eyestreated with the vehicle without TTX (control) (mean+/-SD; N=6).

FIG. 8 is a chart showing corneal anesthesia in abraded rabbit eyesfollowing topical application of 20 microliters of 0.1 mM TTX. Cornealsensation was tested for TTX-treated and vehicle treated (control) eyesat 4 and 6 hours after a single topical administration of TTX or thecontrol. The chart shows that a single 20 ul dose of 0.1 mM TTX, whenapplied to abraded rabbit corneas, resulted in 6 hours of localanesthesia as measured by the blink response.

FIG. 9 is a chart showing corneal anesthesia in abraded rabbit eyesfollowing topical application of 20 microliters of 0.2 mM TTX. Cornealsensation was tested for TTX-treated and vehicle treated (control) eyesat 4, 6 and 8 hours after a single topical administration of TTX or thecontrol. The chart shows that a single 20 ul dose of 0.2 mM TTX, whenapplied to abraded rabbit corneas, results in 8 hours of localanesthesia as measured by the blink response.

FIG. 10 is a chart showing corneal sensitivity after topicaladministration of a single dose of either 1 mM STX or vehicle (control)to abraded rabbit corneas as measured by blink response at 2, 4 and 6hours after administration.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The following terms, as referred to herein, have the following meanings,unless otherwise indicated.

"Long-acting sodium channel blocking compound" refers to a compound,e.g. a toxin or analog that, when administered to a mammal in aneffective concentration, causes local anesthesia lasting at least 3 to10 hours, and specifically binds to the extracellular mouth of thesodium channel, occluding the channel by a mechanism separate from thatof local anesthetics, such as lidocaine, proparacaine. See J. F.Butterworth and G. R. Strichartz, Anesthes. 72:711-734 (1990).Long-acting sodium channel blocking compounds, when administered in asingle dose, may effect local anesthesia of long duration, lasting atleast 3 hours (3 to 10 hours), preferably at least 4 hours (4-10 hours),and most preferably at least 6 to 10 hours. Such long-acting sodiumchannel blocking compounds include compounds that specifically bind to asite formed in part by an extracellular region of the alpha subunit of asodium channel. See Goodman & Gilman's, The Pharmacological Basis ofTherapeutics, Ninth Edition 340-341 (1996); H. Terlau, et al., Fed.Europ. Biochem. Soc. 293(1-2): 93-96 (1991); Encyclopedia of MolecularBiology, pages1127-1131 (ed. J. Kendrew 1994). Examples of long-actingsodium channel blocking compounds that bind to an extracellular siteformed by the SS1 and SS2 segments of the alpha subunit include but arenot limited to tetrodotoxin, saxitoxin, chiriquitoxin, GTTX (from G.tamarensis), gonyautoxins (GTX-I-V, GTX-I, GTX-II, GTX-III),neosaxitoxin, and derivatives and analogs thereof. D. J. Bower, et al.,Clinical Toxicology, 18(7):813-863 (1981). Examples of long-actingsodium channel blocking compounds that bind to an extracellular siteformed by the SS3 and SS4 segments of the alpha subunit, include but arenot limited to alpha-scorpion toxin and sea anemone toxin. See Rogers,J. C. et al., J. Biol. Chem. 271(27):15950-15962 (1996).

"Saxitoxin" or "STX" refers to a compound comprising a tetrahydropurinemoiety composed of two guanidine units fused together in a stableazaketal linkage, having a molecular formula C₁₀ H₂₇ N₇ O₄.2--HCl, (mol.wt. 299.30) and to derivatives thereof, including but not limited tohydroxysaxitoxins and neosaxitoxin. Bower et al., NonproteinNeurotoxins, Clin. Toxicol. 18(7):813-863 (1981).

"Tetrodotoxin" or "TTX" refers to the amino perhydroquinazoline compoundhaving the molecular formula C₁₁ H₁₇ N₃ O₈ and to derivatives thereof,including but not limited toanhydrotetrodotoxin, tetrodaminotoxin,methoxytetrodotoxin, ethoxytetrodotoxin, deoxytetrodotoxin andtetrodonic acid (Kao). Examples of TTX analogs include novel TTX analogsisolated from other organisms, as well as those that are partially ortotally chemically synthesized. See e.g., Yotsu, M. et al. Agric. Biol.Chem., 53(3):893-895 (1989). Such analogs bind to the same site on thealpha subunit of sodium channels as does TTX.

"Anesthesia" refers to the loss of sensation, and, as used hereinencompasses analgesia, the reduction in perceived pain withoutnecessarily loss of sensation.

"Topical administration or topically administering" refers toapplication to a tissue of a mammal, including but not limited toapplication to epithelial tissue which has been damaged, lost orde-epithelialized.

"Epithelial tissue region" refers to an area of epithelial tissue in amammal, where the epithelial layer is intact, damaged, or partially orcompletely absent.

"Pharmaceutically acceptable dose" refers to administration of an amountof a long-acting sodium channel blocking compound effective to achieve alocal anesthetic effect for a clinically useful period of time.

"Opthalmically acceptable dose" refers to administration of an amount ofa long-acting sodium channel blocking compound effective to achieve alocal anesthetic effect in an eye for a clinically useful period oftime.

II. Administration of Long-acting Sodium Channel Blocking Compounds

The invention is directed to a method of providing local anesthesia to amammal experiencing pain in a tissue, preferrably an epithelial tissueregion. The method includes topically administering to the region, aneffective dose of a long-acting sodium channel blocking compound in asuitable vehicle, including an opthalmically suitable vehicle.

In general embodiments of the invention, described in more detail below,the method provides local anesthesia to a patient having pain in anepithelial tissue region associated with damage or loss of epithelialtissue as a result of, for example, plastic surgery, canker sores,burns, sore throats, genital lesions, upper or lower gastrointestinalbronchoscopy or endoscopy, intubation, dermatologic abrasions orchemical skin peels.

In one preferred embodiment, the method is for producing localanesthesia in de-epithelialized corneal tissue in the eye of a patientafter injury to the eye, for example, photorefractive keratectomy, bytopically administering to the eye an effective dose of a long-actingsodium channel blocking compound in a suitable vehicle.

In experiments performed in support of the present invention, the use oftetrodotoxin as a topical anesthetic was demonstrated by administrationto corneal epithelial tissue in rabbit eyes. Tetrodotoxin wasadministered to healthy rabbit eyes and to de-epithelialized rabbit eyesto determine the extent and duration of local anesthesia provided bytetrodotoxin and to evaluate its toxicity, as will be described below.

A. Administration in Healthy Rabbit Eyes

1. Administration of 0.1 mM, 1 mM and 10 mM tetrodotoxin

The extent and duration of local anesthesia provided by tetrodotoxin wasdetermined by administration of the anesthetic at concentrations of 0.1mM, 1 mM and 10 mM to healthy rabbit eyes. As described in Example 1,tetrodotoxin was administered to the right eye of 18 rabbits byinstillation of 40 μl of the drug in a sodium citrate vehicle. Thefellow, left eye of each rabbit received 40 μl of the sodium citratevehicle as a control.

The anesthetic effect was determined by the corneal blink test, asdescribed in Example 1. In this test, the cornea of each rabbit wasmechanically stimulated and the rabbit's response was scored on a scaleof 1-3, where a score of 1 was assigned for no blink, a score of 2 wasassigned for a partial blink without full lid closure and a score of 3was assigned for a full blink. Each eye was scored prior toadministration of the drug and at intervals after administration for 8hours.

FIG. 1A shows the results of administration of tetrodotoxin, where theresponse score is plotted as a function of time after administration of0.1 mM (open circles), 1 mM (open diamonds) and 10 mM (open squares)tetrodotoxin. At a concentration of 0.1 mM tetrodotoxin, no localanesthesia was produced in the rabbits, as evidenced by the responsescores of 2.8-3.0.

The 1 mM tetrodotoxin formulation produced local anesthesia in all ofthe animals, as evidenced by the one minute score of 1.17. The localanesthesia effect was relatively short-lived, as evidenced by the 1 hourscore of 1.5 and by the 3 hour score of 2.83.

The 10 mM tetrodotoxin formulation produced longer-lasting localanesthesia. At one minute after administration, all rabbit corneaswerelocally anesthetic with a mean anesthesia score of 1.0 (SD=0). At 4hours, local anesthesia was still present with a mean score of 1.17(SD=0.4 1), and by 8 hours, the mean score of 2.0 indicated residuallocal anesthesia in most animals. As late as 8 hours, 5 of 6 rabbitsshowed some residual local anesthesia.

FIG. 1B shows the corneal response scores following administration ofthe placebo control vehicle. As seen, corneal blink response remained ata score of about 3, indicating that none of the eyes treated with thecontrol vehicle had diminished corneal sensitivity.

2. Comparative Test

In a separate experiment (Example 1B), the anesthetic duration providedby tetrodotoxin (10 mM and 1 mM) was compared to that provided by 0.5%proparacaine, a common topical ocular anesthetic (Rosenwasser).Following the procedure set forth in Example 1, the right eye of each ofthe 18 test rabbits received 40 μl of the test drug with the fellow,left eye of each rabbit receiving a placebo control. The results areshown in FIGS. 2A and 2B.

As seen in FIG. 2A, the 10 mM tetrodotoxin dose (open squares) producedsignificantly longer local anesthesia than proparacaine (open circles).While proparacaine produced local anesthesia in 6 of 6 rabbits at 1minute (Table 2), by 1 hour the mean blink response score had increasedto 2.50 (SD+0.84), and at 3 hours, all eyes receiving proparacaine hadnormal sensation. In contrast, as late as 5 hours, 4 of 6 rabbitsreceiving tetrodotoxin showed some residual local anesthesia with a meanscore of 1.83, which was significantly different (p=0.0325, Wilcoxontest) from the mean score of 3.00 obtained with proparacaine at 5 hours.

3. Slit Lamp Examination, Pachometry, Toxicity

To assess whether administration of tetrodotoxin causes clinicalalterations in the cornea, the eyes of animals treated with 10 mM and 1mM tetrodotoxin were examined at 12 and 24 hours after drugadministration by slit lamp biomicroscopy with a portable Kowa slitlamp, with and without fluorescein stain from impregnated stripsmoistened with balance salt solution. There was no apparent ocularirritation or epithelial toxicity after administration, nor was thereany obvious discomfort, as evidenced by prolonged eye closure orrepetitive blinking.

To evaluate whether endothelial function was significantly affected byadministration of tetrodotoxin, pachometry (Humphrey Pachometer)readings prior to and 24 hours after tetrodotoxin administration wereperformed on animals treated with 10 mM and 1 mM tetrodotoxin.Pachometry readings on rabbit eyes receiving the highest doses oftetrodotoxin showed no evidence of corneal thickening during the 24 hourobservation period, as shown in Table 3 in Example 1B.

With respect to systemic toxicity, each test rabbit was observedcarefully for any signs of systemic toxicity during the 24 hour testperiod. No rabbit had any alterations of feeding, movement, respiration,or alertness during this period that might suggest a toxic effect oftetrodotoxin. No rabbit died or was noted to have abnormalities inbehavior.

Collectively, and in summary, these experiments demonstrate thattetrodotoxin, administered topically by instillation of drops into theeye, provides an anesthetic effect. The dosage administered from the 10mM tetrodotoxin formulation achieved corneal anesthesia with a rapidonset and anesthetic duration for nearly 4 hours, with some anestheticeffect, as evidenced by reduced corneal sensation, provided for at least8 hours, a significant improvement over that provided by proparacaine.The tests also showed a dose response effect, with lower doses producingeither shorter or no anesthetic effect. Importantly, there was noapparent signs of ocular irritation, corneal thickening or systemictoxicity after administration of tetrodotoxin.

B. Administration in De-epithelialized Rabbit Eyes

In other experiments performed in support of the invention, tetrodotoxinwas administered to de-epithelialized rabbit corneas. As set forth inExample 2A, a central epithelial corneal abrasion was created in eacheye and tetrodotoxin, at a concentration of 0.1 mM, 1 mM or 10 mM, wasadministered into the inferior conjunctival cul-de-sac of one eye. Acontrol vehicle was administered into the fellow eye.

Corneal sensation was tested, as described in Example 2A, and therabbit's response was scored as described above on a scale of 1-3, witha score of 3 indicating full responsiveness and a score of 1 indicatingfull local anesthesia. Corneal sensation was tested prior toadministration of tetrodotoxin and after administration at 2, 4, 6 and 8hours (Example 2B).

FIG. 3A shows corneal blink response in centrally de-epithelializedrabbit corneas treated with topical 0.01 mM (open circles), 0.1 mM (opendiamonds) or 1.0 mM tetrodotoxin (open squares). FIG. 3B shows thecorneal blink response for the fellow, left eyes treated with thevehicle only control.

As seen in FIG. 3A, tetrodotoxin induced local anesthesia ofde-epithelialized corneas varied as a function of dose. At 2 hours aftertetrodotoxin application, all of the tetrodotoxin concentrations showedsome anesthetic effect. At 4 hours, rabbit eyes that were treated with1.0 mM and 0.1 mM tetrodotoxin were significantly different, with meananesthesia scores of 1.00 (SDD=0.00, P=0.0011) and 1.67 (SD=0.52,P=0.0011), respectively. At 6 hours after tetrodotoxin administration, 5of 6 rabbit eyes treated with 1.0 mM tetrodotoxin were anesthetic with amean response score of 1.50 (SD=0.84, P=0.0076). By 8 hours the meanresponse score for 1.0 mM tetrodotoxin was 2.50 (SD=0.84), with 2 of 6rabbits showing significantly reduced sensation.

The rabbits were observed for changes in feeding habits, movement,respiration and alertness for 24 hours, with no apparent changesobserved.

The effectiveness of tetrodotoxin produced by repeated dosing wasdetermined, as described in Example 2C. Tetrodotoxin at a concentrationof 1 mM was administered every 6 hours to the centrallyde-epithelialized cornea of six rabbits for 24 hours. Corneal sensationwas measured every 3 hours for 30 hours.

The results, plotted in FIG. 4, show that at 3 hours after the firstdose of tetrodotoxin (at t=0) all of the rabbit eyes were anestheticwith a mean response score of 1.00 (SD=0.00, P=0.0011). All six rabbiteyes remained anesthetic for the duration of the experiment, with the 40μl dose of 1 mM tetrodotoxin administered every six hours as indicatedby the arrows in the figure. The mean response scores range from 1.00 to1.17 (P=0.0011) over the 24 hour period of administration. At 6 hoursafter the final dose of tetrodotoxin (30 hours after the initial dose),6 of 6 rabbit eyes were still anesthetic with a mean response score of1.17 (SD=0.41, P=0.0011). At 10 hours after the final dose (34 hoursafter the initial dose), 5 of 6 rabbit eyes had normal sensation with amean response score of 2.83 (SD=0.41).

The results presented in FIGS. 3 and 4 show that tetrodotoxin is aneffective anesthetic at a dosage provided by the 1 mM formulation inpartially de-epithelialized corneas. The dose required for effectivelocal anesthesia is significantly reduced in de-epithelialized tissuescompared to intact corneas, as evidenced by comparing FIG. 1A with FIG.3. The effective dose administered from the 1 mM formulation in rabbitsis approximately 1% of the lethal human dose, as discussed in moredetail below.

The studies also show that tetrodotoxin administered every 6 hours for24 hours from formulation having a 1 mM concentration of tetrodotoxinproduced local anesthesia for greater than 30 hours and that, at thisdosing frequency, a reduction in corneal sensation was observed for 30hours.

C. Re-epithelialization of Tissue

As discussed above, ocular pain after a photorefractive keratectomyprocedure is severe for 24-48 hours and characterized by throbbing,watering and foreign body sensations. Conventional treatment includestopical application of non-steroidal anti-inflammatory drugs, low dosetopical anesthetics, oral analgesics or bandage soft contact lens. SeeR. Lim-Bon-Siong, et al., Efficacy and Safety of the ProTek (Vifilcon A)Therapeutic Soft Contact Lens After Photorefractive Keratectomy, Am. J.Opthalmology, 125:169-176 (1998). It has been shown that frequentlyapplied topical anesthetics inhibit corneal re-epithelialization(Rosenwasser).

To determine the effect of tetrodotoxin on corneal re-epithelialization,1 mM tetrodotoxin was administered to rabbit corneas having anepithelial defect. The epithelial defect was created in the right eye of12 rabbits using n-heptanol soaked filter paper discs, as described inExample 3. The rabbits were randomized into two test groups fortreatment with 40 μl of 1 mM tetrodotoxin every 8 hours or for notreatment. The size of the defect was measured in both test groups atregular intervals after creation of the defect. The results are shown inFIG. 5.

As seen in FIG. 5, corneal defects of corneas left untreated and ofcorneas treated with tetrodotoxin were essentially identical in theirrates of healing. By 49 hours, 4 of 6 untreated and 3 of 6tetrodotoxin-treated corneas were completely healed. At 56 hours, 4 of 6untreated and 4 of 6 tetrodotoxin-treated corneas were completelyhealed. By 66 hours, all but one cornea from each group had completelyhealed.

To determine if tetrodotoxin treatment had altered corneal thickness,pachometry was performed 90 hours after the initial wound was created.There was no statistical difference in corneal thickening betweenuntreated and tetrodotoxin-treated corneas.

This experiment shows that topical tetrodotoxin administered every 8hours had no effect on corneal re-epithelialization compared tountreated control rabbit eyes. The rate of healing was nearly identicalfor the two groups, and tetrodotoxin had no apparent effect on cornealthickness compared to untreated control eyes.

III. Method of Administration

As discussed above, the present invention provides a method of producinglocal anesthesia in a subject experiencing pain in an epithelial tissueregion, typically an epithelial tissue region that is damaged or ispartially or completely absent, by administering a therapeuticallyeffective dose of tetrodotoxin.

Tetrodotoxin is a nonprotein neurotoxin which is found in multiplediverse animal species, including puffer fish, porcupine fish, gobyfish, newts, frogs and the blue-ringed octopus (Bower). Tetrodotoxin andits anesthetic properties have been reviewed (Blakenship, Kao, Ogura).

In the experiments described above, tetrodotoxin was administeredtopically to the cornea of rabbit eyes and found to provide long-actingpain relief with no signs of systemic or local toxicity.

More generally, the method of the present invention is intended for usein providing local anesthesia for pain associated with any epithelialtissue region in a subject, for example, pain associated with epithelialulcers, such as a canker sore or genital lesions. Canker sores can occuralone or in groups on the inside of the cheek or lip or underneath thetongue. Severely affected people have continuously recurring ulcerswhich last for one to two weeks (Clayman). Long-acting, topicalpainkillers, such as tetrodotoxin, can ease the pain withoutsignificantly retarding healing of these oral lesions.

Genital ulcers are usually caused by sexually transmitted diseases,including herpes and syphilis. The early stages of syphilis arecharacterized by a hard chancre, a painful ulcer where bacteria haspenetrated the skin. This may be followed by shallow, elongated ulcersonce the chancre has healed. Such ulcers are painful. Genital ulcerationmay also be a side effect of drugs taken orally or caused by solutionsapplied to genital warts. Tetrodotoxin administered in accordance withthe method of the invention provides relief from the pain associatedwith such ulcers.

Pain in epithelial tissue is also caused by burns. Burns affecting theepidermal layer are usually associated with pain, restlessness andfever. Treatment of such a burn in accordance with the method of theinvention can provide relief from the attendant pain, while allowingepithelial healing.

Pain as a result of damage to or loss of epithelial tissue is alsoassociated with other conditions and procedures, such as sore throatsand plastic surgery, for example carbon dioxide laser surgery to removefor skin resurfacing and removal of wrinkles (Rosenberg), burns, genitallesions, upper or lower gastrointestinal bronchoscopy or endoscopy,intubation, dermatologic abrasions or chemical skin peels. Tetrodotoxinadministered in accordance with the method of the invention isbeneficial in relieving pain associated with such damaged tissueswithout significantly retarding healing.

A. Tetrodotoxin and Saxitoxin Formulations and Dosages

For use in the eye, tetrodotoxin and saxitoxin are typicallyadministered in an aqueous solution, which may be an aqueous suspension,an ointment, a gel or an aqueous polymer solution. Preferably, thesolution has a pH of between 4-8, more preferably between about 5-7.5.Such ophthalmic formulations suitable for topical and intraocularadministration can be formulated in accordance with techniques known tothose of skill in the art.

Typically, the active ingredient tetrodotoxin or saxitoxin is formulatedinto purified water or a physiological saline solution as a majorvehicle. However, it will be appreciated that the ophthalmic formulationcan contain other components, including, but not restricted to,buffering means to maintain or adjust pH, such as acetate buffers,citrate buffers, phosphate buffers and borate buffers; viscosityincreasing agents such as polyvinyl alcohol, celluloses, such ashydroxypropyl methyl cellulose and carbomer; preservatives, such asbenzalkonium chloride, chlorobutanol, phenylmercuric acetate and phenylmercuric nitrate; tonicity adjustors, such as sodium chloride, mannitoland glycerine; and penetration enhancers, such as glycols, oleic acid,alkyl amines and the like. The addition of a vasoconstrictor to theformulation is also contemplated. Combination formulations comprisingsaid long-acting sodium channel blocking compound and an antibiotic, asteroidal or a non-steroidal anti-inflammatory drug are alsocontemplated.

The ophthalmic formulation may also be a suspension of particles, suchas polymer particles or liposomes, for entrapping the active compound.For example, polymer particles prepared by suspension or emulsionpolymerization to have a particle size of below about 50 μm, forentrapping the active compound or for increasing viscosity can be addedto the formulation.

The final ophthalmic formulations in unit dosage form are preferablystored in opaque or brown containers to protect then from lightexposure. Multi-dose containers can also be used, if desired,particularly if the final formulation has a low viscosity to permitconstant, accurate dosages to be administered dropwise to the eye.

The method of the invention, more generally, provides local anesthesiato mucous membranes or damaged, e.g., abraded, skin by topicaladministration of tetrodotoxin or saxitoxin in the form of solutions,creams, ointments, gels, aerosols or the like. Such formulations areprepared using ingredients and according to procedures known to those ofskill in the art or as described in, for example, REMINGTON'SPHARMACEUTICAL SCIENCES.

Administration of tetrodotoxin has been well studied in a number ofanimal species (Kao), and the lethal oral dose in humans has beenestimated to be about 10-18 μg/kg (Kao). For a 70 kg person, the lethaldose would therefore be 0.7-1.26 mg.

In the experiments performed in support of the present invention,described above, a 40 μl aliquot of 0.01 mM, 1 mM or 10 mM tetrodotoxinwas administered topically to rabbits' eyes. This corresponds to adosage of between 0.127-127 μg of tetrodotoxin, well below the lethaloral human dose and giving a sufficient safety margin to allow for anydifferences in systemic absorption between topical and oraladministration. In additional experiments, 20 μl aliquots of 0.1 mM and0.2 mM tetrodotoxin were administered topically to partiallyde-epithelialized rabbit eyes, resulting in 6 and 8 hours of localanesthesia respectively.

For administration to the eye, the tetrodotoxin formulation, as aconcentration of between 0.001-10 mM and in the form of aqueoussolution, suspension, ointment or the like, is administered dropwise tothe eye. A single drop typically administers between 10-50 μl. The dropvolume and solution concentration, of course, determine the dosagedelivered, which, for the ranges specified here, is between about0.003-160 μg.

Saxitoxin (1 mM) was administered in a 20 μl dose to partiallyde-epithelialized rabbit corneas, resulting in 4 hours of localanesthesia. This dosage of saxitoxin corresponds to approximately 5.9 μgof saxitoxin, well below the estimated human lethal dose (oral) ofbetween 300 μg to 1.0 mg. See Bower et al., Clin. Toxicol.,18(7):813-863 (1981).

For administration to the eye, the saxitoxin formulation, as aconcentration of between 0.001-10 mM and in the form of aqueoussolution, suspension, ointment or the like, is administered dropwise tothe eye. A single drop typically administers between 10-50 μl. The dropvolume and solution concentration, of course, determine the dosagedelivered, which, for the ranges specified here, is between about 0.003to 149 μg.

It will be appreciated that the dose and concentration of tetrodotoxinor saxitoxin administered is determined on an individual basis, withconsideration given to such factors as age and body weight of thepatient, as well as to the route of administration and the clinicalanaesthetic requirements.

Preferably, tetrodotoxin or saxitoxin is administered topically to thepainful epithelial tissue region by application of a formulation havinga tetrodotoxin or saxitoxin concentration of between about 0.001-10 mM.The actual dosage of tetrodotoxin or saxitoxin administered will, ofcourse, depend on the amount of formulation applied and the surface areaover which it is applied.

The dosing regimen is selected to provide pain relief as needed for aparticular clinical condition. Often, pain is most intense in the first24-72 hours following damage to the tissue, by a surgical procedure orother trauma, and administration every 6 hours during this time periodis effective to provide a long-acting anesthetic effect. This wasdemonstrated in the experiment discussed above where tetrodotoxin wasadministered every 6 hours for 24 hours to achieve greater than 30 hoursof local anesthesia in rabbits, with no signs of irritation or toxicity.

It will be appreciated that topical administration of eithertetrodotoxin or saxitoxin may be combined with conventional modes ofpain relief, such as administration of oral or topical non-steroidalanti-inflammatory drugs or antibiotics and the use of bandage softcontact lens in ocular applications. For example, methods of theinvention include applying to the corneal surface of an eye of a mammal,a bandage contact lens, wherein said lens is capable of delivering anopthalmically effective dose of said long-acting sodium channel blockingcompound to said corneal surface. One of skill in the art willappreciate that the there are a number of ocular drug delivery systemsand methods that can be used with the present invention, some of whichare described in Lee, V. and Robinson, J. R., Review: Topical OcularDrug Delivery: Recent Developments and Future Challenges, J. OcularPharmacol. 2(1):67-108 (1986) (incorporated herein by reference).

Moreover, another preferred method of producing anesthesia in an eye ofa mammal comprises topically administering to the corneal surface of aneye of a mammal an opthalmically effective dose of proparacaine or othercomparable local anesthetic, including tetracaine or benoxinate, in anopthalmically acceptable vehicle before administering to said eye, anopthalmically effective dose of a long-acting sodium channel blockingcompound in an opthalmically acceptable vehicle. Prior administration ofproparacaine or other comparable local anesthetic, to the eye of amammal effectively diminishes the production of tears by the eye,preparing the eye for topical administration of a long-acting sodiumchannel blocking compound, such as TTX or STX. By decreasing tearing andanesthetizing the corneal surface of an eye, the prior administration ofproparacaine, tetracaine or benoxinate, decreases the amount of thelong-acting sodium channel blocking compound that would wash away withtears. Thus, the method enables administration of lower effective dosesof the long-acting sodium channel blocking compound. For example,dosages of proparacaine range from 0.05% to 0.5%, and preferably, from0.05 to 0.1% formulations in any opthalmically acceptable vehicle.

From the foregoing, it can be appreciated how various features andobjects of the invention are met. The method of the invention providesan effective, long-lasting local anesthesia by topical administration ofa long-acting sodium channel blocking compound to a painful epithelialtissue region. The studies reported herein illustrate, using a rabbitmodel, that a single dose of tetrodotoxin at a concentration of 1 mM or10 mM achieves local anesthesia with rapid onset and for at least 8hours. A repeated dosing regimen is effective to produce localanesthesia for greater than 30 hours. Importantly, no evidence of eitherocular or systemic toxicity in the test animals was observed at any ofthe dosage levels, including 10 mM tetrodotoxin. Moreover, a single doseof saxitoxin at a concentration of 1 mM achieves local anesthesia uponadministration to a partially abraded cornea with rapid onset and for atleast 4 hours.

One of skill in the art will appreciate that any of the long-actingsodium channel blocking compounds can be used according to the methodsand procedures described herein to determine pharmaceutically oropthalmically effective doses and other aspects of the invention.

VI. EXAMPLES

The following examples illustrate the methods and compositions of theinvention, but are in no way intended to limit the invention.

Materials: Tetrodotoxin was purchased from Sigma Chemical Co. (St.Louis, Mo.) in vials containing 1 mg tetrodotoxin and approximately 5 mgsodium citrate buffer, pH=4.3, in lyophilized form.

Saxitoxin and additional tetrodotoxin was purchased from AlexisChemicals.

Example 1 Administration of Tetrodotoxin to Healthy Rabbit Eyes

A. 0.1 mM, 1 mM and 10 mM Tetrodotoxin

Tetrodotoxin formulations of 0.1 mM, 1 mM and 10 mM were prepared in a60 mM sodium citrate carrier at pH 4.3.

Eighteen New Zealand white rabbits were divided into three groups of sixrabbits. Each rabbit received a 40 μl aliquot of one of the tetrodotoxinformulations into the inferior conjunctival cul-de-sac of the right eye,with the left fellow eye receiving 40 μl of the pH 4.3 60 mM sodiumcitrate carrier as a control.

Corneal sensation was tested with a 4-0 silk suture mounted upon awooden cotton tip applicator such that the suture extended 5 mm beyondthe wooden end of the applicator. The cornea was mechanically stimulatedcentrally three times with the suture to produce grossly visibleindentation of the cornea as the endpoint (similar to previous rabbitmodel of corneal anesthesia reported in Maurice D M, Singh T., Theabsence of corneal toxicity with low-level topical anesthesia. Am JOphthamol. 99:691-696. (1985)). Care was taken not to stimulate theeyelashes. The rabbit's response was graded in the following fashion: noblink=1; partial blink without full eyelid closure=2; fuill blink=3.Thus, a score of 3 indicates full responsiveness and a score of 1indicates full local anesthesia. The highest anesthesia score of the 3tests was recorded for each time point.

Corneal sensation was tested prior to administration of drugs and againat 1 minute, 1 hour, 4 hours and 8 hours. The results are tabulated inTable 1 below and shown in FIGS. 1A-1B. Statistical analysis was by thenon-parametric Wilcoxon test with a statistical sugnificance of p-<0.05.

                                      TABLE 1                                     __________________________________________________________________________             Mean Blink Response (N = 6)                                          Drug Treatment                                                                         0 min.                                                                              1 min.                                                                              60 min.                                                                             240 min.                                                                            480 min.                                     __________________________________________________________________________    0.1 mM tetrodotoxin                                                                    2.83 (0.41)                                                                         2.67 (0.52)                                                                         2.67 (0.82)                                                                         3.00 (0.00)                                                                         2.83 (0.41)                                  0.1 mM         3.00 (0.00)1)                                                                         3.00 (0.00)                                                                       3.00 (0.00)                                                                           3.00 (0.00)                                tetrodotoxin/control                                                          1 mM tetrodotoxin                                                                         2.83 (0.41)                                                                       1.17 (0.41)                                                                           1.50 (0.84)                                                                       2.83 (0.41)                                                                           2.83 (0.41)                               1 mM           2.83 (0.41)41)                                                                        2.83 (0.41)                                                                       3.00 (0.00)                                                                           2.67 (0.52)                                tetrodotoxin/control                                                          10 mM tetrodotoxin                                                                       3.00 (0.00)                                                                       1.00 (0.00)                                                                           1.00 (0.00)                                                                       1.17 (0.41)                                                                           2.00 (0.89)                                10 mM          3.00 (0.00)00)                                                                        2.83 (0.41)                                                                       3.00 (0.00)                                                                           3.00 (0.00)                                tetrodotoxin/control                                                          __________________________________________________________________________     *numbers in parenthesis are standard deviation of mean blink response for     n = 6.                                                                   

B. Comparative Study: 1 mM, 10 mM Tetrodotoxin and Proparacaine

Eighteen New Zealand white rabbits were divided into three groups of sixrabbits. Each rabbit received a 40 μl aliquot into the inferiorconjunctival cul-de-sac of the right eye of 1 mM or 10 mM tetrodotoxinor of proparacaine 0.5% (Ophthetic). The left fellow eye received 40 μlof a placebo, control vehicle (60 mM, pH 4.3 sodium citrate).

Following the procedure described in Example 1A, the anesthetic durationwas determined by measuring corneal blink response in the test rabbitsat 0 min, 1 minute, 1 hour, 3 hours and 5 hours. The results aretabulated in Table 2 and shown in FIGS. 2A-2B.

                                      TABLE 2                                     __________________________________________________________________________             Mean BIink Response (N = 6)                                          Drug Treatment                                                                         0 min.                                                                              1 min.                                                                              60 min.                                                                             180 min.                                                                            300 min.                                     __________________________________________________________________________    Proparacaine                                                                           3.00 (0.00)                                                                         1.00 (0.00)                                                                         2.50 (0.84)                                                                         3.00 (0.00)                                                                         3.00 (0.00)                                  Proparacaine/control                                                                   3.00 (0.00)                                                                         3.00 (0.00)                                                                          2.50 (0.84)                                                                         3.00 (0.00)                                                                          3.00 (0.00)                                1 mM tetrodotoxin                                                                        3.00 (0.00)                                                                       2.17 (0.75)                                                                          2.00 (0.89)                                                                         2.50 (0.55)                                                                          2.83 (0:41)                                1 mM           3.00 (0.00)0.00)                                                                     2.83 (0.41)                                                                         3.00 (0.00)                                                                          3.00 (0.00)                                tetrodotoxin/control                                                          10 mM tetrodotoxin                                                                        3.00 (0.00)                                                                      1.00 (0.00)                                                                          1.00 (0.00)                                                                         1.33 (0.82)                                                                          1.83 (0.98)                                10 mM          3.00 (0.00).00)                                                                      3.00 (0.00)                                                                         3.00 (0.00)                                                                          3.00 (0.00)                                tetrodotoxin/control                                                          __________________________________________________________________________     *numbers in parenthesis are standard deviation of mean blink response for     n = 6.                                                                   

The extent and duration of local anesthesia after topical TTX wasadministered to intact rabbit corneas varied as a function of dose (FIG.1A, Table 1). At 0.1 mM TTX, only partial local anesthesia was producedin 2 of 6 rabbits. At 1 mM TTX, local anesthesia initially was producedin 6 of 6 animals but the effect was generally short-lived. At oneminute after administration of TTX, the mean anesthesia score was 1.17(SD=0.41). At 1 hour the score was 1.50 (SD=0.84) and by 3 hours themean score had increased to 2.83 (SD=0.41). At 6 hours the TTX-vehiclehad a better anesthetic score than the 1 mM TTX treated eyes. However,neither of these scores were significantly different from each other orfrom the pretreated eyes anesthesia score of 3.0. (SD=0).

At 10 mM, TTX produced a more reproducible and longer lasting localanesthesia. At one minute after administration, all rabbit corneas wereanesthetic with a mean anesthesia score of 1.00 (SD=0). At 4 hours,local anesthesia was still present with a mean score of 1.17 (SD=0.41).As late as 8 hours, 4 of 6 rabbits showed some residual local anesthesiawith a mean score of 2.00 (SD=0.89). This was significantly different,(P=0.0325), from the mean score of 3.00 (SD=0) obtained with vehiclealone at 8 hours (Table 1).

The anesthetic duration of 10 mM TTX was compared to that of 1 mM TTX,and proparacaine (FIG. 2, Table 2). At the 10 mM dose, TTX producedsignificantly longer local anesthesia than proparacaine. Whileproparacaine produced local anesthesia in 6 of 6 rabbits at 1 minute, by1 hour the mean score had increased to 2.50 (SD=0.84), and at 3 hours,all eyes receiving proparacaine had normal sensation. As late as 5hours, 4 of 6 rabbits receiving TTX showed some residual localanesthesia with a mean score of 1.83 (SD=0.98). This was significantlydifferent, (P=0.0325), from the mean score of 3.00 (SD=0) obtained withproparacaine or vehicle alone at 5 hours (Table 2).

C. Pachometry

To evaluate whether endothelial function was significantly affected byadministration of 10 mM or 1 mM TTX, pachometry readings were made(Humphrey Pachometer, Humphrey Instr., San Leandro, Calif.) prior to and24 hours after TTX administration. The results are given in Table 3 asmean corneal thickness, with the standard deviation indicated inparenthesis. Pachometry readings on rabbits receiving the highest dosesof TTX showed no evidence of corneal thickening during the 24 hourobservation period.

                  TABLE 3                                                         ______________________________________                                        Drug             Corneal Thickness (mm)                                       Treatment        0 hrs.    24 hrs.                                            ______________________________________                                        proparacaine     0.38 (0.03)                                                                             0.38 (0.04)                                        proparacame/control                                                                                      0.39 (0.04)                                                                         0.38 (0.03)                                  1 mM tetrodotoxin                                                                                              0.39 (0.04)                                  1 mM tetrodotoxin/control                                                                           0.36 (0.01)                                                                              0.39 (0.04)                                  10 mM tetrodotoxin                                                                                             0.37 (0.01)                                  1 mM tetrodotoxin/control                                                                           0.36 (0.01)                                                                              0.38 (0.04)                                  ______________________________________                                         *numbers in parenthesis are standard deviation of mean corneal thickness      for n = 6.                                                               

Drug treatments labelled "/control" represent fellow eyes of eitherproparacaine or TTX-treated eyes, wherein the fellow eyes were treatedwith the citrate vehicle alone.

Slit Lamp Examination and Fluorescein Staining.

Slit lamp biomicroscopy with a portable slit lamp (Kowa SL-5, KowaCompany, Japan) was performed with and without fluorescein stain fromimpregnated strips moistened with balance salt solution at 12 and 24hours after topical administration.

To assess whether TTX administration caused clinical alterations in thecornea, all animals were examined with a slit lamp after fluoresceinstaining. Despite the acidic vehicle, TTX administration did not causeany apparent ocular irritation after administration. There was noobvious discomfort in any of the rabbits evidenced by prolonged eyeclosure or repetitive blinking. No ocular injection was noted during the24 hour observation period. At 3 hours most of the rabbits had a mildcentral punctuate epithelial keratopathy in the area of cornealsensation testing, but there was no difference between rabbits receivingproparacaine and TTX. By 24 hours, all signs of epithelial damage haddisappeared by slit lamp examination and fluorescein staining.

Toxicity

The rabbits were observed for changes in feeding habits, movement,respiration and alertness during the first 24 hours by the experimentersand for the subsequent week daily by animal care personnel.

No rabbit had any alterations of feeding, movement, respiration, oralertness during this period that suggested a toxic effect of the TTX.No rabbit died or was noted to have abnormalities in behavior by theanimal care personnel for 7 days subsequent to TTX administration.

Example 2 Topical Administration of Tetrodotoxin to PartiallyDe-epithelialized Rabbit Corneas

A. Corneal Abradement and Blink Response Test

After general anesthesia and topical application of 0.5% proparacaine toeach eye, a #69 Beaver blade was used to create a central epithelialdefect which measured between 3.0-3.5 mm diameter in both eyes of eachtest rabbit.

Corneal sensation was tested with a 4-0 silk suture mounted upon awooden cotton tip applicator such that the suture extended 5 mm beyondthe wooden end of the applicator. Because the central cornea was oftenrendered largely anesthetic following mechanical epithelial debridement,the cornea was stimulated in the mid-peripheral cornea, outside of theabraded area, with the suture to produce grossly visible indentation ofthe cornea as the endpoint. Care was taken not to stimulate theeyelashes. The rabbit's response was graded in the following fashion; noblink=1, partial blink without full eyelid closure=2, full blink=3.

B. Dose Response

Tetrodotoxin in a pH 4.3 sodium citrate vehicle was formulated intoconcentrations of 1 mM, 0.1 mM, and 0.01 mM.

New Zealand white rabbits were divided into three experimental groups ofsix. Each rabbit received a 40 μl aliquot of tetrodotoxin at aconcentration of 0.01 mM, 0.1 mM or 1 mM into the inferior conjunctivalcul-de-sac of the right eye. A 40 μl of a pH 4.3 sodium citrate vehicleas a control into the fellow, left eye.

Corneal sensation was tested prior to administration of tetrodotoxin andafter administration at 2, 4, 6 and 8 hours. The results, presented asthe mean score for the 6 rabbits in each test group, are tabulated belowin Table 4 and shown in FIGS. 3A-3B, where FIG. 3A shows the responsescores for eyes treated with tetrodotoxin and FIG. 3B shows the responsescores for the fellow, control-vehicle treated eyes. Statisticalanalysis using the Wilcoxon test was performed by comparing thetetrodotoxin treated eyes to the pre-operative anesthesia responsescore.

                  TABLE 4                                                         ______________________________________                                        Drug     Mean Blink Response (N = 6)                                          Treatment                                                                              2 hrs.    4 hrs.    6 hrs.  8 hrs                                    ______________________________________                                        1.0 mM   1.00 (0.00)                                                                             1.00 (0.00)                                                                             1.50 (0.84)                                                                           2.50 (0.84)                              tetrodotoxin                                                                  1.0 mM       2.83 (0.41)                                                                         3.00 (0.00)                                                                              3.00 (0.00)                                                                           3.00 (0.00)                             tetrodotoxin/                                                                 control                                                                       0.1 mM          1.17 (0.41)                                                                       1.67 (0.52)                                                                              2.83 (0.41)                                                                           3.00 (0.00)                            tetrodotoxin                                                                  0.1 mM       2.83 (0.41)                                                                         3.00 (0.00)                                                                              3.00 (0.00)                                                                           3.00 (0.00)                             tetrodotoxin/                                                                 control                                                                       0.01 mM      1.50 (0.84)                                                                         2.33 (0.82)                                                                              2.83 (0.41)                                                                           2.67 (0.52)                             tetrodotoxin                                                                  0.01 mM     2.50 (0.84)                                                                          2.67 (0.52)                                                                              3.00 (0.00)                                                                           3.00 (0.00)                             tetrodotoxin/                                                                 control                                                                       ______________________________________                                         *numbers in parenthesis are standard deviation of mean blink response for     n = 6.                                                                   

TTX-induced local anesthesia of de-epithelialized corneas varied as afunction of dose. At 2 h after TTX application, all of the TTXconcentrations showed some anesthetic effect. TTX at 1.0 mM, 0.1 mM, and0.01 mM had mean anesthesia scores of 1.00 (SD=0.00, P=0.0011), 1.17(SD=0.41, P=0.0076), and 1.50 (SD=0.84, P=0.0076) respectively. At 4 h,rabbit eyes that were treated with 1.0 mM and 0.1 mM TTX were stillsignificantly anesthetic with mean anesthesia scores of 1.00 (SD=0.00,P=0.0011) and 1.67 (SD=0.52, P=0.0011), respectively. In contrast, themean anesthesia score of rabbits treated with 0.01 mM TTX was returningto normal at 2.33 (SD=0.82) by 4 h. By 6 h, rabbit eyes treated witheither 0.1 mM or 0.01 mM had mean scores of 2.83 (SD=0.41). At 6 h afterTTX administration, five of six rabbit eyes treated with 1.0 mM TTX werestill partially anesthetic with a mean anesthesia score of 1.50(SD=0.84, P=0.0076). By 8 h the mean anesthesia score for 1.0 mM TTX wasapproaching normal at 2.50 (SD=0.84), with only two of six rabbitsshowing any anesthetic effect.

C. Dosing Frequency

Experiments were conducted to test whether TIX could produce prolongedeffectiveness with repeated dosing. A 40 μl aliquot of 1 mM tetrodotoxinwas administered every 6 hours for 24 hours to the centrallyde-epithelialized cornea of 6 rabbits. Corneal sensation was monitored,by scoring the eye for blink response as described above, every 3 hoursfor 24 hours and at 17 hours, 30 hours and 34 hours after the initialdose. Response scores after administration of tetrodotoxin were comparedto pre-operative response scores for statiscal analysis by the Wilcoxontest. The results are tabulated in Table 5 and shown in FIG. 4, wherethe arrows in the figure indicate the dosage times.

                  TABLE 5                                                         ______________________________________                                        Time (hours)  Blink Response                                                  ______________________________________                                                                    3.00 (0.00)                                        3                                    1.00 (0.00)                              6                                    1.17 (0.41)                              9                                    1.00 (0.00)                             12                                   1.00 (0.00)                              15                                   1.00 (0.00)                              18                                   1.00 (0.00)                              21                                   1.17 (0.41)                              24                                   1.00 (0.00)                              17                                   1.00 (0.00)                              30                                   1.17 (0.41)                              34                                   2.83 (0.41)                              ______________________________________                                         *numbers in parenthesis are standard deviation of mean blink response for     n = 6.                                                                   

At 3 hours after the first administration, all of the rabbit eyes wereanesthetic with a mean anesthesia score of 1.00 (SD=0.00, P=0.0011). Allsix rabbit eyes remained anesthetic for the duration of the experiment,with mean anesthesia scores ranging from 1.00 to 1.17 (P=0.0011throughout). At 6 h after the final TTX administration and 30 h afterthe initial TTX administration, six of six rabbit eyes were stillanesthetic with a mean anesthesia score of 1.17 (SD=0.41, P=0.0011). At10 h after the final TTX administration and 34 h after the initial TTXadministration, five of six rabbit eyes had normal sensation with a meananesthesia score of 2.83 (SD=0.41).

Example 3 Administration of Tetrodotoxin to Partially De-epithelializedRabbit Corneas

A. Corneal Abradement

To determine whether TTX inhibits corneal re-epithelialization, twelveNew Zealand White rabbits were anesthetized with a mixture of keamineand xylazine. A corneal trephine was used to make Whatman #2 filterpaper discs measuring 7.5 mm in diameter. The discs were soaked inn-heptanol solution and blotted to remove excess liquid. One disc wasplaced on the central cornea of the right eye of each rabbit for 30seconds. After removal, the cornea was washed with balanced saltsolution to remove the loosened epithelial cells. Topical fluoresceinwas applied and the diameter of the epithelial defect was measured in 2meridians (12:00-6:00 and 3:00-9:00) using calipers. The radius of thedefect was determined, and the defect area was calculated as previouslydescribed using an equation that corrects for the curvature of therabbit cornea. Crosson C E, Klyce S D, Beuerman R W (1986) Epithelialwound closure in the rabbit cornea. Invest Ophthalmol Vis Sci27:464-473.

B. Effect of Multiple Doses of TTX on Re-epithelialization of AbradedRabbit Corneas

The rabbits were randomized into two test groups for treatment with 40μl of 1 mM tetrodotoxin every 8 hours or for no treatment. The rabbitswere re-anesthetized with keratin and xylazine and epithelial defectsize was re-measured with calipers at 17, 32, 42, 49, 56, and 66 h afterthe creation of epithelial defects. Twenty-four hours after the cornealepithelial defects were closed (completely healed), pachometry (HumphreyUltrasonic Pachometer) was performed in both the experimental and felloweyes of each rabbit.

The results are shown in FIG. 5 and Table 6.

                  TABLE 6                                                         ______________________________________                                        Corneal Defect Size                                                                      AREA 0F DEFECT (mm.sup.2)                                          TIME (h)     CONTROL    TTX                                                   ______________________________________                                           0                   53.0 +/- 2.0                                                                   49.0 +/- 5.8                                          17                               37.9 +/- 7.1                                 32                               15.7 +/- 7.1                                 42                                5.6 +/- 6.3                                 49                                3.0 +/- 5.7                                 56                                2.5 +/- 5.9                                 66                                0.3 +/- 0.7                                 ______________________________________                                    

Corneal defects of untreated and 1.0 mM TTX-treated corneas wereessentially identical in their healing rates. By 48 h, four of sixuntreated corneas and three of six TTX-treated corneas were completelyhealed. At 56 h, four of six untreated and four of six TTX-treatedcorneas were completely healed. By 66 h, all but one cornea from eachgroup had completely healed. In these remaining eyes, only a smalldefect, 0.67+/-1.63 mm2 for the TTX-treated rabbit and 0.29+/-0.72 mm2for the untreated control rabbit, remained

C. Effect of Multiple Doses of TTX on Corneal Thickness afterRe-epithelialization

In order to determine whether TTX had an effect on corneal thicknessafter re-epithelialization compared to untreated control eyes, cornealthickness was measured 92 hours after creation of a corneal abrasion(OD) in rabbit eyes that had been treated with multiple doses of 1.0 mMTTX. Corneal thickness of healed corneal abrasions (OD) and uninjuredfellow eyes (OS) were measured by pachometry. The results are given asthe mean defect area and the standard deviation (n=6)

                  TABLE 7                                                         ______________________________________                                        DRUG        CORNEAL THICKNESS                                                 TREATMENT         OD                        OS                                ______________________________________                                        NONE        0.39 +/- 0.04   0.43 +/- 0.04                                     1.0 mM TTX         0.40 +/- 0.04                                                                                 0.41 +/- 0.05                              ______________________________________                                    

There was no statistically significant difference in corneal thicknessbetween untreated and TTX-treated corneas (Table 7).

Example 4 Topical Administration of Tetrodotoxin to Rabbit Eyes afterExcimer Laser Keratectomy

TTX in a pH 4.3 sodium citrate vehicle was used in the followingexperiments (Sigma Chemical Co., St. Louis, Mo.). New Zealand whiterabbits were divided into two experimental groups, each consisting ofsix rabbits. After general anesthesia by intramuscular injection of amixture of xylazine and keratin, followed by topical application of 0.5%proparacaine (Ophthetic, Allergan, Irvine, Calif.) to each right eye,the rabbits underwent excimer laser keratectomy on their right eyes.

Excimer laser keratectomy was performed using the Star Excimer LaserSystem (VISX, Inc.) in phototherapeutic keratectomy (PTK) mode. Excimerlaser keratectomy was performed to create a 5 mm diameter wound, 75 μmin depth. The repetition rate of the laser was set at 6 Hz with a pulseenergy density of 160 mJ/cm2.

One group of six rabbits then received a 40 μl aliquot of 1 mM TTX intothe inferior conjunctival cul-de-sac of their right eyes and the othergroup of six rabbits received 40 μl of the pH 4.3 sodium citrate vehicleinto the inferior conjunctival cul-de-sac of their right eyes as acontrol. The rabbits were treated with 1 mM TTX or vehicle again at 6,12, 18, and 24 hours.

Corneal sensation was tested as previously described. Briefly, sensationwas tested with a 5 mm silk suture mounted on a wooden cotton tipapplicator. The cornea was stimulated with the suture in themid-peripheral cornea, outside of the excimer laser keratectomy treatedarea. Care was taken not to stimulate the eyelashes. The rabbit'sresponse was graded in the following fashion: no blink=1, partial blinkwithout full eyelid closure=2, full blink=3. Corneal sensation wastested at 3, 6, 9, 12, 15 ,18, 21, 24, 30, 32, and 40 hours. At 6, 12,18 and 24 hours the corneal sensation was tested prior tore-administration of TTX or vehicle. The rabbits were observed forchanges in feeding habits, movement, respiration and alertness. The dataare presented in FIG. 6 as the mean score of 6 rabbits/treatment group.TTX treated eyes' anesthesia scores were compared to vehicle treatedeyes' anesthesia scores for statistical significance by the Wilcoxontest.

Administration of 40 μl of 1 mM TTX every 6 hours for 24 hours afterexcimer laser keratectomy produced nearly complete local anesthesia forat least 30 hours with a mean anesthesia score of 1.17 (SD=0.41)(p=0.011, Wilcoxon test) (FIG. 6). Three hours following eachapplication of TTX, the mean anesthesia score for TTX treated eyes was1.0 (SD=0). Six hours following each application of TTX the meananesthesia scores were between 1.0 (SD=0) and 1.5 (SD=0.84). At least4/6 of the rabbits were completely anesthetic at six hours followingeach application of TTX. At 32 hours, 8 hours following the fmalapplication of TTX, there was still significant local anesthesia of theTTX treated corneas (p=0.0325, Wilcoxon test). The mean anesthesia scorewas 2.0 (SD=0.89). At 40 hours, 18 hours following the final applicationof TTX, all of the rabbits' corneas had returned to normal sensation. Incontrast, after 9 hours, vehicle treated eyes all had normal sensationfor the duration of the experiment (FIG. 6). There was a very slightlocal anesthesia of the vehicle treated eyes at 6 and 9 hours followingexcimer laser keratectomy, with the mean anesthesia score being 2.83(SD=0.41). However, this was due in each case to 1/6 rabbits scoring 2,5/6 rabbits scoring 3, and was not significant. At 3 hours followingexcimer laser keratectomy the vehicle treated eye's mean anesthesiascore was 2.16 (SD=0.75). This was probably due to lingering effects ofthe general and topical anesthetics that were administered prior to theexcimer laser keratectomy procedure.

Example 5 Effect of Topical Administration of TTX Following PRK onCorneal Re-epithelialization

To determine whether TTX inhibited corneal re-epithelializationfollowing excimer laser keratectomy, topical fluorescein was appliedafter rabbits were given general anesthesia by intramuscular injectionof a mixture of xylazine and keratin. The diameter of the circularepithelial defect was measured using calipers in 2 meridians (12:00-6:00and 3:00-9:00), and the radius of the defect was calculated. The radiuswas used to calculate the area of the defect using the method of Crossonet al. that corrects for the curvature of the rabbit cornea (Crosson CE, et al., Invest Ophthalmol Vis Sci., 27:464-473 (1986)). Generalanesthesia was administered and the size of the epithelial defect wasmeasured at 24, 40, 49, 63, 68, and 72 hours following excimer laserkeratectomy. Observers were masked as to the contents of eye drops givento the rabbits.

To assess whether or not repeated administration of TTX had any effecton the rate of epithelial healing, epithelial defect area was measuredover 72 hours following excimer laser keratectomy. As shown in FIG. 7,there was little difference in healing rate of vehicle and TTX treatedeyes. At 24 hours there was no significant difference in healing betweenTTX treated and vehicle treated eyes (p>0.05, t-test). At 40 hours theTTX treated eyes had larger defects than vehicle treated eyes 7.85 mm2vs. 4.54 mm2 (p<0.025, t-test). However, at 49 hours, and thereafter,both groups were equally healed (p>0.05, t-test).

Toxicity.

The rabbits involved in the corneal anesthesia and corneal wound healingexperiments following excimer laser keratectomy described above wereobserved carefully for any signs of systemic toxicity during the courseof the experiment. No rabbit had any alterations of feeding, movement,respiration, or alertness during this period that suggested a toxiceffect of the TTX. No rabbit died or was noted to have abnormalities inbehavior subsequent to TTX administration. Additionally, no signs oflocal toxicity such as ocular injection or corneal haze were apparent inany rabbit.

Example 6 Systemic Absorption of TTX Topically Applied to Abraded RabbitEyes

TTX (Alexis Corporation) was formulated into 20 μl doses containing 100μg of m in a pH 4.3 citrate buffer (60 mM). Four Dutch banded rabbitswere weighed. After general anesthesia by intramuscular injection of amixture of xylazine and keratin, followed by topical application of 0.5%proparacaine to each eye, a #69 Beaver blade was used to create acentral epithelial defect which measured between 4 mm diameter in oneeye of each rabbit. Each rabbit then received a 20 μl aliquot of TTXinto the inferior conjunctival cul-de-sac of the abraded eye. At 10 min,20 min and 40 min following administration of TTX, 4 ml of blood wastaken from each rabbit. The blood was clotted and the serum collected.

TTX was purified from serum samples using the purification scheme ofYasumoto and Michishita. Fluorometric determination of tetrodotoxin byhigh performance liquid chromatography. Agric Biol Chem 49: 3077-3080(1985). Briefly, 300 μl of each serum sample was extracted by boilingfor 10 min in 0.02N acetic acid, and TTX was purified by chromatographyover Amberlite CG-50. TTX was eluted from the Amberlite with 0.5 Nacetic acid, samples were dried in a vacuum centrifuge and TTX wasresuspended in 50 μl of phosphate buffered saline.

Assays to quantify the amount of TTX purified from serum samples wereperformed using a tissue culture bioassay. Hamasaki, K, Kogure, K andOhwada, K. A biological method for the quantitative measurement oftetrodotoxin (TTX): tissue culture bioassay in combination with awater-soluble tetrazolium salt. Toxicon 34: 490-495 (1996). Briefly, 2to 3×105 mouse Neuro 2A cells per well were plated on 96 well microtiterplates in 200 μl of RPMI-1640 medium containing 10 fetal bovine serumand incubated overnight in a 37° C. tissue culture incubator. After 24hours the cells were given 10 μl of the TTX samples, 20 μl of 10 mMouabain, and 20 μl of 0.5 mM veratridine. After an additional 24 hoursat 37° C., the cells were given 20 μl of WST-1 cell counting reagent(Dojindo Corp.) and allowed to incubate an additional 3 hours at 37° C.The yellow color produced by the WST-1 reagent was quantified by readingthe absorbance at 430 nm with a reference wave length of 600 nm. Allassays were performed in duplicate. TTX concentrations were derived bycomparison to a standard curve generated using normal rabbit serum thathad been spiked with known amounts of pure TTX and subjected to the samepurification scheme as the rabbit serum samples. The results are givenin Table 8 as the average amount of TTX in μg per ml of serum at eachtime point in the table +/- standard deviation (N=4) (SE=StandardError). The average amount of TTX absorbed into the serum is derived byassuming a serum volume equal to 3% of the mass of each rabbit. Kaplan,H M and Timmons, E H. The Rabbit: a Model of the Principals of MammalianPhysiology and Surgery. Academic Press, NY (1979).

                  TABLE 8                                                         ______________________________________                                                                   TTX in Serum                                       Time                       (Total μg                                       (mins)                                                                                TTX (μg/ml)                                                                            Std. Error                                                                                absorbed)                                                                               Std. Error                          ______________________________________                                         0    0                    0                                                  10            0.16 +/- 0.09                                                                      (SE = 0.04)                                                                              6.39 +/- 4.61                                                                             (SE = 2.30)                         20            0.33 +/- 0.16                                                                      (SE = 0.08)                                                                                14.44 +/- 6.09                                                                         (SE = 3.04)                          40            0.31 +/- 0.11                                                                     (SE = 0.06)                                                                                  13.92 +/- 4.06                                                                         (SE = 2.03)                         ______________________________________                                    

Thus, after 20 minutes about 14% of the applied dose of TTX was absorbedinto the serum. This persisted for at least 40 minutes.

Example 7 Topical Application of Single Dose (20 μl) of 0.1 mM TTX toAbraded Rabbit Cornea

TTX (Alexis Corporation) was obtained and formulated into a 0.1 mMconcentration in a pH 4.3 sodium citrate vehicle (60 mM). Dutch bandedrabbits were divided into two experimental groups of five and six. Aftergeneral anesthesia by intramuscular injection of a mixture of xylazineand keratin, followed by topical application of 0.5% proparacaine toeach eye, a #69 Beaver blade was used to create a central epithelialdefect which measured between 4 mm diameter in both eyes of each rabbit.Five rabbits received a 20 μl aliquot of 0.1 mM TTX into one eye and sixrabbits received 20 μl of the pH 4.3 sodium citrate vehicle as a controlinto one eye. Experimenters were masked to the contents of each aliquot.

Corneal sensation was tested with a 4-0 silk suture mounted upon awooden cotton tip applicator such that the suture extended 5 mm beyondthe wooden end of the applicator. Because the central cornea was oftenrendered anesthetic following mechanical epithelial debridement, thecornea was stimulated in the mid-peripheral cornea, outside of theabraded area, with the suture to produce grossly visible indentation ofthe cornea as the endpoint. Care was taken not to stimulate theeyelashes. The rabbit's response was graded in the following fashion; noblink=1, partial blink without full eyelid closure=2, full blink=3. Ascore of 3 indicates full responsiveness and a score of 1 indicates fulllocal anesthesia. Corneal sensation was tested prior to administrationof drugs and again at 4 and 6 hours. The rabbits were observed forchanges in feeding habits, movement, respiration and alertness for 24hours. The data are presented as the mean score of 6 rabbits/treatmentgroup. TTX treated eyes' anesthesia scores were compared to theirvehicle treated fellow eyes' anesthesia scores for statistical analysisby the Wilcoxon test. The results are shown in FIG. 8.

At 4 hours the vehicle treated eyes had an average anesthesia score of2.67 (SD=0.52). In contrast the TTX treated eyes were significantlyanesthetic with a mean score of 1.20 (SD=0.45) (p=0.0411). At 6 hoursthe vehicle treated eyes had a mean anesthesia score of 3.00 (SD=0.00).In contrast at 6 hours the TTX treated eyes were still significantlyanesthetic with a mean anesthesia score of 2.00 (SD=1.00) (p=0.0411).Thus, one 20 μl dose of 0.1 mM TTX provided at least 6 hours of localanesthesia when applied to an abraded rabbit cornea.

Example 8 Topical Application of Single Dose (20 μl) of 0.2 mM TTX toAbraded Rabbit Cornea

TTX was obtained and formulated into a 0.2 mM concentration in a pH 4.3sodium citrate vehicle (60 mM). Dutch banded rabbits were divided intotwo experimental groups of five and six. After general anesthesia byintramuscular injection of a mixture of xylazine and keratin, followedby topical application of 0.5% proparacaine to each eye, a #69 Beaverblade was used to create a central epithelial defect which measuredbetween 4 mm diameter in both eyes of each rabbit. Six rabbits receiveda 20 μl aliquot of 0.2 mM TTX into one eye and five rabbits received 20μl of the pH 4.3 sodium citrate vehicle as a control into one eye.Experimenters were masked to the contents of each aliquot.

Corneal sensation was tested with a 4-0 silk suture mounted upon awooden cotton tip applicator such that the suture extended 5 mm beyondthe wooden end of the applicator. Because the central cornea was oftenrendered anesthetic following mechanical epithelial debridement, thecornea was stimulated in the mid-peripheral cornea, outside of theabraded area, with the suture to produce grossly visible indentation ofthe cornea as the endpoint. Care was taken not to stimulate theeyelashes. The rabbit's response was graded in the following fashion; noblink=1, partial blink without full eyelid closure=2, full blink=3. Ascore of 3 indicates full responsiveness and a score of 1 indicates fullanesthesia. Corneal sensation was tested prior to administration ofdrugs and again at 4, 6, and 8 hours. The rabbits were observed forchanges in feeding habits, movement, respiration and alertness for 24hours. The data are presented as the mean score of 6 rabbits/treatmentgroup. TTX treated eyes' anesthesia scores were compared to theirvehicle treated fellow eyes' anesthesia scores for statistical analysisby the Wilcoxon test. The results are shown in FIG. 9

All of the vehicle treated eyes were normal throughout the experimentalperiod with an average anesthsia score of 3.00 (SD=0.00). In contrastthe TTX treated eyes showed significant local anesthesia. At 4 hours theTTX treated eyes were significantly anesthetic with a mean score of 1.17(SD=0.41) (p=0.0022). At 6 hours the TTX treated eyes were stillsignificantly anesthetic with a mean anesthesia score of 1.33 (SD=0.82)(p<0.0152). At 8 hours the TTX treated eyes were still significantlyanesthetic with a mean anesthesia score of 1.50 (SD=0.84) (p<0.0152).Thus, one 20 μl dose of 0.2 mM TTX provided at least 8 hours of localanesthesia when applied to an abraded rabbit cornea.

Example 9 Calculation of Dosages

TTX has been determined to be an effective and non-toxic anesthetic fortopical administration to partially de-epithelialized rabbit comeas forat least 6 h at a dose of 1.0 mM, or approximately 1.0-1.8% of theestimated lethal human dose. Even lower effective dosages for topicaladministration to partially de-epithelialized rabbit corneas wereestablished, including 20 μl aliquots of either 0.1 mM TTX or 0.2 mMTTX, corresponding to 0.6 μg and 1.27 μg, respectively.

While the toxic dose of TTX has been well studied in a number of animalspecies (Kao CY, Tetrodotoxin, saxitoxin and their significance in thestudy of excitation phenomena. Pharm Rev 18:997-1049 (1966)), it hasbeen only imprecisely estimated for humans. Cornish reported a case ofTTX intoxication and estimated the lethal oral dose to be about 10-18μg/kg based on the amount of fish tissue consumed (Cornish J N,Susceptibility of man to pufferfish toxin. Med J Aust 2:48 (1973)). Fora 70-kg person, the lethal dose would therefore be 0.7-1.26 mg. The doselethality curve for TTX is very steep. In mice, the minimumintraperitoneal lethal dose is 8 μg/kg and the LD100 is 12 μg/kg (KaoCY, Fuhrman F A, Pharmacological studies on tarichatoxin, a potentneurotoxin. J Pharmacol 140:31-40 (1963)). The lethal dose for oraladministration in mice is much higher, 332 μg/kg (Kao CY, Pharm Rev18:997-1049 (1966)). Because of the steep dose-lethality curve,reduction of the effective topical dose of TTX to 1% or less of theminimum lethal dose should result in a therapeutically useful dose.

More sensitive testing systems using an esthesiometer, such as theBoberg-Ans or the Cochet and Bonnet device are well-known in the fieldand can be used to determine safe human dosages. Such testing can helpdetermine whether TTX can produce partial local anesthesia or analgesiaat doses even lower than 1.0-1.8% of the estimated lethal human doseused in this study.

Example 10 Topical Administration of Saxitoxin (STX) to PartiallyDe-epithelialized Rabbit Corneas

Saxitoxin (Alexis Corporation) was formulated into 1 mM concentration inbalanced saline solution (BSS). Ten Dutch banded rabbits were dividedinto two experimental groups of five. After general anesthesia byintramuscular injection of a mixture of xylazine and keratin, followedby topical application of 0.5% proparacaine to each eye, a #69 Beaverblade was used to create a central epithelial defect which measuredbetween 4 mm diameter in one eye of each rabbit. Five rabbits thenreceived a 20 μl aliquot of Saxitoxin into the inferior conjunctivalcul-de-sac of one eye and five rabbits received a 20 μl aliquot of theBSS vehicle as a control into the fellow eye. Experimenters were maskedto the contents of each aliquot.

Corneal sensation was tested with a 4-0 silk suture mounted upon awooden cotton tip applicator such that the suture extended 5 mm beyondthe wooden end of the applicator. Because the central cornea was oftenrendered anesthetic following mechanical epithelial debridement, thecornea was stimulated in the mid-peripheral cornea, outside of theabraded area, with the suture to produce grossly visible indentation ofthe cornea as the endpoint. Care was taken not to stimulate theeyelashes. The rabbit's response was graded in the following fashion; noblink=1, partial blink without full eyelid closure=2, full blink=3. Ascore of 3 indicates full responsiveness and a score of 1 indicates fulllocal anesthesia. Corneal sensation was tested prior to administrationof drugs and again at, 2, 4, and 6 hours. The rabbits were observed forchanges in feeding habits, movement, respiration and alertness for 24hours. The data are presented as the mean score of 5 rabbits/treatmentgroup. The results are shown in FIG. 10 (As the mean local anesthesiascore +/- standard error) and again in Table 9 (as the mean anesthesiascore +/- the standard deviation). Statistical analysis was by theWilcoxon test.

                  TABLE 9                                                         ______________________________________                                        Time (min)   1 mM Saxitoxin                                                                            Vehicle                                              ______________________________________                                         120         1.0 +/- 0.00                                                                              2.8 +/- 0.45                                         240                     1.80 +/- 1.09                                                                      2.8 +/- 0.45                                     360                     3.0 +/- 0.00                                                                        2.8 +/- 0.45                                    ______________________________________                                    

After 2 hours all of the Saxitoxin treated rabbit eyes weresignificantly anesthetic compared to vehicle treated eyes with a meananesthesia score of 1.0 (SD=90)(p=0.004). In contrast the vehicletreated eyes had an mean anesthesia score of 2.8 (SD=0.45). After 4hours the Saxitoxin treated eyes were still significantly anestheticwith a mean anesthesia score of 1.8 (SD=1.09)(p=0.0159), while thevehicle treated eyes had a mean anesthesia score of 2.8 (SD=0.45). By 6hours the Saxitoxin treated eyes had returned to normal with a meananesthesia score of 3.0 (SD=0), while the vehicle treated eyes had amean anesthesia score of 2.8 (SD=0.45). None of the rabbits showed anysigns of systemic toxicity. Thus, treatment of corneal abraded rabbitcorneas with 20 μl of 1 mM Saxitoxin provided at least 4 hours ofcorneal anesthesia.

References

Blakenship, J. E., Perspectives in Biology and Medicine, summer:509-526(1976).

Bower, D. J., et al., Clinical Toxicology, 18(7), 813-863 (1981).

Cherry, P. M., et al., Supplement to Ophthalmic Surgery and Lasers,25(5), S477-80 (1996).

Clayman, C. B., "THE AMERICAN MEDICAL ASSOCIATION ENCYCLOPEDIA OFMEDICINE", Random House, New York, (1989).

Goto, T. et al., Tetrodotoxin, Tetrahedron, 21:2-59-2088 (1965).

Hamasaki K. et al., A Biological Method for the Quantitative Measurementof Tetrodotoxin (TTX): Tissue Culture Bioassay in Combination with aWater-Soluble Tetrazolium Salt, Toxicon. 34(4):490-495 (1996).

Lee, V. and Robinson, J. R., Review: Topical Ocular Drug Delivery:Recent Developments and Future Challenges, J. Ocular Pharmacol.2(1):67-108 (1986).

Kao, C. Y., Pharmaceutical Reviews, 18(2), 997-1049 (1966).

Ogura, Y. and Mori, Y., European Journal of Pharmacology, 3:58-67(1968).

Salminen L., Review: Systemic Absorption of Topically Applied OcularDrugs in Humans, J. Ocular Pharmacol., 6(3):243-249 (1990).

Schwartz, D. M. et al., Experimental Use of Tetrodotoxin for CornealPain After Excimer Laser Keratectomy, Cornea 17(2):196-199 (1998).

Tutton M. K., et al., J. Cataract Refract Surg. 22(5), 536-541 (1996).

Rosenberg, G. J. et al., Clinics in Plastic Surgery, 23(1):29 (1996).

Rosenwasser, G. O. D., Intl. Ophthalmology Clinics, 29(3), 153-158(1989).

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the invention has been described with respect to particularembodiments, it will be apparent to one of ordinary skill in the artthat many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the invention.

We claim:
 1. A method of producing local anesthesia in a partially orcompletely de-epithelialized tissue region of a mammal,comprisingtopically administering an anesthetically effective dose of apharmaceutical composition consisting essentially of a long-actingsodium channel blocking compound, in a pharmaceutically suitablevehicle, to said de-epithelialized tissue region of said mammal, whereinsaid de-epithelialized tissue region is a corneal region, a region inthe upper or lower gastrointestinal tract, or a genital lesion in thegenital area.
 2. The method of claim 1, wherein said long-acting sodiumchannel blocking compound does not inhibit re-epithelialization of saidepithelial tissue region.
 3. The method of claim 2, wherein said sodiumchannel blocking compound is administered every 6-8 hours for betweenabout 24-72 hours.
 4. The method of claim 2, wherein said long-actingsodium channel blocking compound is a compound capable of specificallybinding to a site on an extracellular region of a sodium channel alphasubunit.
 5. The method of claim 4, wherein said site is on an SS2extracellular region of a sodium channel alpha subunit.
 6. The method ofclaim 5, wherein said long-acting sodium channel blocking compound istetrodotoxin.
 7. The method of claim 6, wherein said effective dose oftetrodotoxin is administered from a formulation containing tetrodotoxinat a concentration of between 0.001-10 mM.
 8. The method of claim 6,wherein said tetrodotoxin is administered in a vehicle having a pH ofbetween 4-8.
 9. The method of claim 8, wherein said vehicle has a pH ofbetween 5-7.5.
 10. The method of claim 5, wherein said long-actingsodium channel blocking compound is saxitoxin.
 11. The method of claim1, wherein said administering is to a partially or completelyde-epithelialized comeal tissue region.
 12. The method of claim 1,wherein said administering comprises instilling drops of said sodiumchannel blocking compound to the eye following corneal surgery.
 13. Themethod of claim 1, wherein said epithelial tissue region is a region inthe upper or lower gastrointestinal tract.
 14. The method of claim 1,wherein said epithelial tissue region is associated with genital lesionsin the genital area.
 15. A method of producing local anesthesia in aneye of a mammal, comprisingtopically administering to the cornealsurface of the eye of said mammal, in an ophthalmically suitablevehicle, an anesthetically effective dose of a pharmaceuticalcomposition consisting essentially of a long-acting sodium channelblocking compound, said corneal surface having an epithelial layer thatis partially or completely de-epithelialized.
 16. The method of claim15, wherein said long-acting sodium channel blocking compound is acompound capable of specifically binding to a site on an extracellularregion of a sodium channel alpha subunit, wherein said site is on eitheran SS1 region or an SS2 region.
 17. The method of claim 16, wherein saidlong-acting sodium channel blocking compound is tetrodotoxin and saideffective dose is administered from a formulation containingtetrodotoxin at a concentration between about 0.001-10 mM.
 18. Themethod of claim 17, wherein said long-acting sodium channel blockingcompound is tetrodotoxin and said effective dose is administered from aformulation containing tetrodotoxin at a concentration between about0.01 mM to 0.2 mnM.
 19. The method of claim 18, wherein saidtetrodotoxin is administered in a vehicle having a pH of between about4-8.
 20. A method of reducing pain in a mammal following cornealrefractive surgery, comprising,topically administering to a partially orcompletely de-epithelialized corneal surface of an eye of said mammal,in an ophthalmically suitable vehicle, a pain reducing effective dose ofa pharmaceutical composition consisting essentially of a long-actingsodium channel blocking compound.
 21. The method of claim 20, furthercomprising the step of instilling drops of a non-steroidalanti-inflammatory compound in the eye of said mammal.
 22. The method ofclaim 20, further comprising administering to said mammal an antibioticor a non-steroidal anti-inflammatory drug.
 23. A method of reducing painin a mammal following corneal refractive surgery, comprisingtopicallyadministering to a corneal surface of an eye of said mammal, in anophthalmically suitable vehicle, a pain reducing effective dose of apharmaceutical composition consisting essentially of a long-actingsodium channel blocking compound, wherein said administering is byapplying to the eye of said mammal a bandage contact lens, wherein saidlens is capable of delivering said long-acting sodium channel blockingcompound to said corneal surface.
 24. As an article of manufacture, anophthalmically acceptable dosage container comprising:(a) apharmaceutical composition consisting essentially of from about 0.003 μgto 160 μg of tetrodotoxin in an ophthalmically acceptable vehicle at pHof between about 4-8.
 25. The article of claim 24, wherein saidcontainer comprises: from about 0.127 μg to 2.54 μg of tetrodotoxin. 26.A local anesthetic composition which consists essentially of ananesthetically effective amount of tetrodotoxin, in a concentration of0.01 mM to 1 mM, in an ophthalmically acceptable vehicle at pH 4-6. 27.The composition of claim 26, wherein said concentration is between 0.01mM and 0.2 mM tetrodotoxin.
 28. The composition of claim 26, whereinsaid concentration is between 0.01 mM and 0.1 mM tetrodotoxin.
 29. Alocal anesthetic composition which consists essentially of ananesthetically effective amount of saxitoxin, in a concentration of 0.1mM to 10 mM, in an ophthalmically acceptable vehicle at pH 4-8.
 30. Alocal anesthetic composition which consists essentially of ananesthetically effective amount of tetrodotoxin, in a concentration of0.1 mM to 10 mM, in an ophthalmically acceptable vehicle at pH 4-6. 31.A method of producing a non-toxic local anesthesia in an epithelialtissue region of a mammal, comprisingtopically administering ananesthetically effective dose of a pharmaceutical composition consistingessentially of a long-acting sodium channel blocking compound, in apharmaceutically suitable vehicle comprising a citrate buffer at pH 4-8,to said epithelial tissue region of said mammal.
 32. The method of claim31, wherein said pH is 4-5.
 33. The method of claim 31, wherein saidanesthesia has a duration of 4-8 hours.
 34. The method of claim 31,wherein said long-acting sodium channel blocking compound does notinhibit re-epithelialization of said epithelial tissue.
 35. A non-toxiclocal anesthetic composition which consists essentially of ananesthetically effective amount of tetrodotoxin, in a concentration of0.01 mM to 10 mM, in an ophtalmically acceptable vehicle comprisingcitrate buffer at pH 4-8.
 36. The composition of claim 35, wherein saidpH is 4-5.
 37. The composition of claim 35, wherein said pH is 4-5. 38.A non-toxic local anesthetic composition which consists essentially ofan anesthetically effective amount of saxitoxin, in a concentration of0.01 mM to 10 mM, in an ophtalmically acceptable vehicle comprisingcitrate buffer at pH 4-8.